Bioluminescente en moleculaire detectie van therapiefalen in gevoelige en geneesmiddel-resistente modellen voor viscerale leishmaniose
21 september 2018
UAntwerpen, Campus Drie Eiken, gebouw O, Auditorium O7 - Universiteitsplein 1 - 2610 Wilrijk (Antwerpen) (route: UAntwerpen, Campus Drie Eiken
16 - 18 uur
Guy Caljon, Louis Maes
Doctoraatsverdediging Eline Eberhardt - Departement Biomedische Wetenschappen
Visceral leishmaniasis (VL) is an important neglected infectious disease, caused by Leishmania parasites and responsible for 300,000 new infections annually and over 20,000 deaths. While chemotherapy is the key control strategy, only a handful of drugs are available with considerable shortcomings. Miltefosine (MIL) is currently the only oral drug and despite frequent treatment failures (TFs), MIL-resistance (MIL-R) remained limited so far with the isolation of only four resistant field isolates all harboring a defective Ros3 - Miltefosine Transporter (MT) complex. This emphasizes that TF and drug resistance are not necessarily linked and that both are key parameters in epidemiological surveys.
In this work two essential strategies were adopted for VL research in animal models and patients: (i) the development of an extremely sensitive real-time PCR (qPCR) and (ii) the generation of bioluminescent Leishmania lines. These tools are needed to assess infection tropism and parasite sanctuary sites, the impact of drug treatment, the development of drug resistance and its effect on parasite fitness.
Sliced-leader (SL) RNA sequences that are attached to the 5’ end of all mature nuclear mRNAs served as target for a novel qPCR assay. While the widely used kDNA qPCR was hampered by its limited species range and frequent false positivity, the SL-RNA assay enabled universal Leishmania detection with high sensitivity in hamster tissues and human blood. To optimize patient blood sampling, different stabilizing reagents were compared. RNA protect cell reagent and DNA/RNA shield provided the most optimal stabilization for both DNA and RNA.
In vivo bioluminescence imaging (BLI) enabled monitoring of parasite burdens in a non-invasive way with a limit of detection similar to that of microscopy. The MIL-R line displayed a severe fitness loss that could be rescued by episomal reconstitution with a wildtype MT, linking the infectivity defects to mutations in the transporter gene and hereby explaining the scarcity of natural MIL-R parasites. Surprisingly, both in vivo MIL-treatment and in vitro MIL-pre-exposure significantly reversed the fitness loss, indicating a drug-dependent phenotype of MIL-R parasites. MIL-exposure also altered parasite morphology suggesting MIL-incorporation into the parasite membrane. Altogether, this emphasizes the importance of resistance profiling prior to drug administration.
Using paromomycin (PMM), it was further shown that BALB/c mice can be used as exploratory model for drug efficacy studies and the underlying mechanisms of TF. The bone marrow was identified a niche for parasite survival, subsequent expansion and re-colonization of other target organs, such as the spleen.